Mammalian IAP gene family, primers, probes and detection methods

ABSTRACT

Disclosed is substantially pure DNA encoding mammalian IAP polypeptides; and methods of using such DNA to express the IAP polypeptides in cells and animals to inhibit apoptosis. Also disclosed are conserved regions characteristic of the IAP family and primer and probes for the identification and isolation of additional IAP genes. In addition, methods for treating diseases and disorders involving apoptosis are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/011,356, filedFeb. 4, 1998 (now pending), which is a U.S. National Phase applicationof PCT/IB/96/01022, filed Aug. 5, 1996, and published in English underPCT article 21(2), which claims benefit from U.S. Ser. No. 08/576,956,filed Dec. 22, 1995 (now U.S. Pat. No. 6,156,535), which is acontinuation-in-part of U.S. Ser. No. 08/511,485, filed Aug. 4, 1995(now U.S. Pat. No. 5,919,912), all of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

The invention relates to apoptosis.

There are two general ways by which cells die. The most easilyrecognized way is by necrosis, which is usually caused by an injury thatis severe enough to disrupt cellular homeostasis. Typically, the cell'sosmotic pressure is disturbed and, consequently, the cell swells andthen ruptures. When the cellular contents are spilled into thesurrounding tissue space, an inflammatory response often ensues.

The second general way by which cells die is referred to as apoptosis,or programmed cell death. Apoptosis often occurs so rapidly that it isdifficult to detect. This may help to explain why the involvement ofapoptosis in a wide spectrum of biological processes has only recentlybeen recognized.

The apoptosis pathway has been highly conserved throughout evolution,and plays a critical role in embryonic development, viral pathogenesis,cancer, autoimmune disorders, and neurodegenerative disease. Forexample, inappropriate apoptosis may cause or contribute to AIDS,Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis(ALS), retinitis pigmentosa and other diseases of the retina,myelodysplastic syndrome (e.g. aplastic anemia), toxin-induced liverdisease, including alcoholism, and ischemic injury (e.g. myocardialinfarction, stroke, and reperfusion injury). Conversely, the failure ofan apoptotic response has been implicated in the development of cancer,particularly follicular lymphoma, p53-mediated carcinomas, andhormone-dependent tumors, in autoimmune disorders, such as lupuserythematosis and multiple sclerosis, and in viral infections, includingthose associated with herpes virus, poxvirus, and adenovirus.

In patients infected with HIV-1, mature CD4⁺ T lymphocytes respond tostimulation from mitogens or super-antigens by undergoing apoptosis.However, the great majority of these cells are not infected with thevirus. Thus, inappropriate antigen-induced apoptosis could beresponsible for the destruction of this vital part of the immune systemin the early stages of HIV infection.

Baculoviruses encode proteins that are termed inhibitors of apoptosisproteins (IAPs) because they inhibit the apoptosis that would otherwiseoccur when insect cells are infected by the virus. These proteins arethought to work in a manner that is independent of other viral proteins.The baculovirus IAP genes include sequences encoding a ring zincfinger-like motif (RZF), which is presumed to be directly involved inDNA binding, and two N-terminal domains that consist of a 70 amino acidrepeat motif termed a BIR domain (Baculovirus IAP Repeat).

SUMMARY OF THE INVENTION

In general, the invention features a substantially pure DNA molecule,such as a genomic, cDNA, or synthetic DNA molecule, that encodes amammalian IAP polypeptide. This DNA may be incorporated into a vector,into a cell, which may be a mammalian, yeast, or bacterial cell, or intoa transgenic animal or embryo thereof. In preferred embodiments, the DNAmolecule is a murine gene (e.g., m-xiap, m-hiap-1, or m-hiap-2) or ahuman gene (e.g., xiap, hiap-1, or hiap-2). In most preferredembodiments the IAP gene is a human IAP gene. In other various preferredembodiments, the cell is a transformed cell. In related aspects, theinvention features a transgenic animal containing a transgene thatencodes an IAP polypeptide that is expressed in or delivered to tissuenormally susceptible to apoptosis, i.e., to a tissue that may be harmedby either the induction or repression of apoptosis. In yet anotheraspect, the invention features DNA encoding fragments of IAPpolypeptides including the BIR domains and the RZF domains providedherein.

In specific embodiments, the invention features DNA sequencessubstantially identical to the DNA sequences shown in FIGS. 1-6, orfragments thereof. In another aspect, the invention also features RNAwhich is encoded by the DNA described herein. Preferably, the RNA ismRNA. In another embodiment the RNA is antisense RNA.

In another aspect, the invention features a substantially purepolypeptide having a sequence substantially identical to one of the IAPamino acid sequences shown in FIGS. 1-6.

In a second aspect, the invention features a substantially pure DNAwhich includes a promoter capable of expressing the IAP gene in a cellsusceptible to apoptosis. In preferred embodiments, the IAP gene isxiap, hiap-1, or hiap-2. Most preferably, the genes are human or mousegenes. The gene encoding HIAP-2 may be the full-length gene, as shown inFIG. 3, or a truncated variant, such as a variant having a deletion ofthe sequence boxed in FIG. 3.

In preferred embodiments, the promoter is the promoter native to an IAPgene. Additionally, transcriptional and translational regulatory regionsare, preferably, those native to an IAP gene. In another aspect, theinvention provides transgenic cell lines and transgenic animals. Thetransgenic cells of the invention are preferably cells that are alteredin their apoptotic response. In preferred embodiments, the transgeniccell is a fibroblast, neuronal cell, a lymphocyte cell, a glial cell, anembryonic stem cell, or an insect cell. Most preferably, the neuron is amotor neuron and the lymphocyte is a CD4⁺ T cell.

In another aspect, the invention features a method of inhibitingapoptosis that involves producing a transgenic cell having a transgeneencoding an IAP polypeptide. The transgene is integrated into the genomeof the cell in a way that allows for expression. Furthermore, the levelof expression in the cell is sufficient to inhibit apoptosis.

In a related aspect, the invention features a transgenic animal,preferably a mammal, more preferably a rodent, and most preferably amouse, having either increased copies of at least one IAP gene insertedinto the genome (mutant or wild-type), or a knockout of at least one IAPgene in the genome. The transgenic animals will express either anincreased or a decreased amount of IAP polypeptide, depending on theconstruct used and the nature of the genomic alteration. For example,utilizing a nucleic acid molecule that encodes all or part of an IAP toengineer a knockout mutation in an IAP gene would generate an animalwith decreased expression of either all or part of the corresponding IAPpolypeptide. In contrast, inserting exogenous copies of all or part ofan IAP gene into the genome, preferably under the control of activeregulatory and promoter elements, would lead to increased expression orthe corresponding IAP polypeptide.

In another aspect, the invention features a method of detecting an IAPgene in a cell by contacting the IAP gene, or a portion thereof (whichis greater than 9 nucleotides, and preferably greater than 18nucleotides in length), with a preparation of genomic DNA from the cell.The IAP gene and the genomic DNA are brought into contact underconditions that allow for hybridization (and therefore, detection) ofDNA sequences in the cell that are at least 50% identical to the DNAencoding HIAP-1, HIAP-2, or XIAP polypeptides.

In another aspect, the invention features a method of producing an LAPpolypeptide. This method involves providing a cell with DNA encoding allor part of an LAP polypeptide (which is positioned for expression in thecell), culturing the cell under conditions that allow for expression ofthe DNA, and isolating the IAP polypeptide. In preferred embodiments,the IAP polypeptide is expressed by DNA that is under the control of aconstitutive or inducible promotor. As described herein, the promotormay be a heterologous promotor.

In another aspect, the invention features substantially pure mammalianIAP polypeptide. Preferably, the polypeptide includes an amino acidsequence that is substantially identical to all, or to a fragment of,the amino acid sequence shown in any one of FIGS. 1-4. Most preferably,the polypeptide is the XIAP, HIAP-1, HIAP-2, M-XIAP, M-HIAP-1, orM-HIAP-2 polypeptide. Fragments including one or more BIR domains (tothe exclusion of the RZF), the RZF domain (to the exclusion of the BIRdomains), and a RZF domain with at least one BIR domain, as providedherein, are also a part of the invention.

In another aspect, the invention features a recombinant mammalianpolypeptide that is capable of modulating apoptosis. The polypeptide mayinclude at least a RZF domain and a BIR domain as defined herein. Inpreferred embodiments, the invention features (a) a substantially purepolypeptide, and (b) an oligonucleotide encoding the polypeptide. Ininstances were the polypeptide includes a RZF domain, the RZF domainwill have a sequence conforming to:Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,where Xaa1 is any amino acid, Xaa2 is Glu or Asp, Xaa3 is Val or Ile(SEQ ID NO: 1); and where the polypeptide includes at least one BIRdomain, the BIR domain will have a sequence conforming to:Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val,where Xaa1 may be any amino acid and Xaa2 may be any amino acid or maybe absent (SEQ ID NO: 2).

In various preferred embodiments the polypeptide has at least two or,more preferably at least three BIR domains, the RZF domain has one ofthe IAP sequences shown in FIG. 6, and the BIR domains are comprised ofBIR domains shown in FIG. 5. In other preferred embodiments the BIRdomains are at the amino terminal end of the protein relative to the RZFdomain, which is at or near the carboxyl terminus of the polypeptide.

In another aspect, the invention features an IAP gene isolated accordingto the method involving: (a) providing a sample of DNA; (b) providing apair of oligonucleotides having sequence homology to a conserved regionof an IAP disease-resistance gene; (c) combining the pair ofoligonucleotides with the cell DNA sample under conditions suitable forpolymerase chain reaction-mediated DNA amplification; and (d) isolatingthe amplified LAP gene or fragment thereof.

In preferred embodiments, the amplification is carried out using areverse-transcription polymerase chain reaction, for example, the RACEmethod. In another aspect, the invention features an IAP gene isolatedaccording to the method involving: (a) providing a preparation of DNA;(b) providing a detectably labelled DNA sequence having homology to aconserved region of an IAP gene; (c) contacting the preparation of DNAwith the detectably-labelled DNA sequence under hybridization conditionsproviding detection of genes having 50% or greater nucleotide sequenceidentity; and (d) identifying an IAP gene by its association with thedetectable label.

In another aspect, the invention features an IAP gene isolated accordingto the method involving: (a) providing a cell sample; (b) introducing bytransformation into the cell sample a candidate IAP gene; (c) expressingthe candidate IAP gene within the cell sample; and (d) determiningwhether the cell sample exhibits an altered apoptotic response, wherebya response identifies an IAP gene.

In another aspect, the invention features a method of identifying an IAPgene in a cell, involving: (a) providing a preparation of cellular DNA(for example, from the human genome or a cDNA library (such as a cDNAlibrary isolated from a cell type which undergoes apoptosis); (b)providing a detectably-labelled DNA sequence (for example, prepared bythe methods of the invention) having homology to a conserved region ofan IAP gene; (c) contacting the preparation of cellular DNA with thedetectably-labelled DNA sequence under hybridization conditionsproviding detection of genes having 50% nucleotide or greater sequenceidentity; and (d) identifying an LAP gene by its association with thedetectable label.

In another aspect, the invention features a method of isolating an LAPgene from a recombinant library, involving: (a) providing a recombinantlibrary; (b) contacting the library with a detectably-labelled genefragment produced according to the PCR method of the invention underhybridization conditions providing detection of genes having 50% orgreater nucleotide sequence identity; and (c) isolating an IAP gene byits association with the detectable label. In another aspect, theinvention features a method of identifying an IAP gene involving: (a)providing a cell tissue sample; (b) introducing by transformation intothe cell sample a candidate IAP gene; (c) expressing the candidate IAPgene within the cell sample; and (d) determining whether the cell sampleexhibits inhibition of apoptosis, whereby a change in (i.e. modulationof) apoptosis identifies an IAP gene. Preferably, the cell sample is acell type that may be assayed for apoptosis (e.g., T cells, B cells,neuronal cells, baculovirus-infected insect cells, glial cells,embryonic stem cells, and fibroblasts). The candidate IAP gene isobtained, for example, from a cDNA expression library, and the responseassayed is the inhibition of apoptosis.

In another aspect, the invention features a method of inhibitingapoptosis in a mammal wherein the method includes: (a) providing DNAencoding at least one IAP polypeptide to a cell that is susceptible toapoptosis; wherein the DNA is integrated into the genome of the cell andis positioned for expression in the cell; and the IAP gene is under thecontrol of regulatory sequences suitable for controlled expression ofthe gene(s); wherein the IAP transgene is expressed at a levelsufficient to inhibit apoptosis relative to a cell lacking the IAPtransgene. The DNA integrated into the genome may encode all or part ofan IAP polypeptide. It may, for example, encode a ring zinc finger andone or more BIR domains. In contrast, it may encode either the ring zincfinger alone, or one or more BIR domains alone. Skilled artisans willappreciate that IAP polypeptides may also be administered directly toinhibit undesirable apoptosis.

In a related aspect, the invention features a method of inhibitingapoptosis by producing a cell that has integrated, into its genome, atransgene that includes the LAP gene, or a fragment thereof. The IAPgene may be placed under the control of a promoter providingconstitutive expression of the IAP gene. Alternatively, the IAPtransgene may be placed under the control of a promoter that allowsexpression of the gene to be regulated by environmental stimuli. Forexample, the IAP gene may be expressed using a tissue-specific or celltype-specific promoter, or by a promoter that is activated by theintroduction of an external signal or agent, such as a chemical signalor agent. In preferred embodiments the cell is a lymphocyte, a neuronalcell, a glial cell, or a fibroblast. In other embodiments, the cell inan HIV-infected human, or in a mammal suffering from a neurodegenerativedisease, an ischemic injury, a toxin-induced liver disease, or amyelodysplastic syndrome.

In a related aspect, the invention provides a method of inhibitingapoptosis in a mammal by providing an apoptosis-inhibiting amount of IAPpolypeptide. The IAP polypeptide may be a full-length polypeptide, or itmay be one of the fragments described herein.

In another aspect, the invention features a purified antibody that bindsspecifically to an IAP family protein. Such an antibody may be used inany standard immunodetection method for the identification of an IAPpolypeptide. Preferably, the antibody binds specifically to XIAP,HIAP-1, or HIAP-2. In various embodiments, the antibody may react withother IAP polypeptides or may be specific for one or a few LAPpolypeptides. The antibody may be a monoclonal or a polyclonal antibody.Preferably, the antibody reacts specifically with only one of the IAPpolypeptides, for example, reacts with murine and human XIAP, but notwith HIAP-1 or HIAP-2 from other mammalian species.

The antibodies of the invention may be prepared by a variety of methods.For example, the IAP polypeptide, or antigenic fragments thereof, can beadministered to an animal in order to induce the production ofpolyclonal antibodies. Alternatively, antibodies used as describedherein may be monoclonal antibodies, which are prepared using hybridomatechnology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler etal., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981). The invention features antibodiesthat specifically bind human or murine IAP polypeptides, or fragmentsthereof. In particular the invention features “neutralizing” antibodies.By “neutralizing” antibodies is meant antibodies that interfere with anyof the biological activities of IAP polypeptides, particularly theability of IAPs to inhibit apoptosis. The neutralizing antibody mayreduce the ability of IAP polypeptides to inhibit polypeptides by,preferably 50%, more preferably by 70, and most preferably by 90% ormore. Any standard assay of apoptosis, including those described herein,may be used to assess neutralizing antibodies.

In addition to intact monoclonal and polyclonal anti-LAP antibodies, theinvention features various genetically engineered antibodies, humanizedantibodies, and antibody fragments, including F(ab′)2, Fab′, Fab, Fv andsFv fragments. Antibodies can be humanized by methods known in the art,e.g., monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals, are also features of the invention (Green et al., NatureGenetics 7:13, 1994).

Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes methods forpreparing single polypeptide chain antibodies. Ward et al. (Nature341:544, 1989) describe the preparation of heavy chain variable domains,which they term “single domain antibodies,” which have highantigen-binding affinities. McCafferty et al. (Nature 348:552, 1990)show that complete antibody V domains can be displayed on the surface offd bacteriophage, that the phage bind specifically to antigen, and thatrare phage (one in a million) can be isolated after affinitychromatography. Boss et al. (U.S. Pat. No. 4,816,397) describe variousmethods for producing immunoglobulins, and immunologically functionalfragments thereof, which include at least the variable domains of theheavy and light chain in a single host cell. Cabilly et al. (U.S. Pat.No. 4,816,567) describe methods for preparing chimeric antibodies.

In another aspect, the invention features a method of identifying acompound that modulates apoptosis. The method includes providing a cellexpressing an IAP polypeptide, contacting the cell with a candidatecompound, and monitoring the expression of an IAP gene. An alteration inthe level of expression of the IAP gene indicates the presence of acompound which modulates apoptosis. The compound may be an inhibitor oran enhancer of apoptosis. In various preferred embodiments, the cell isa fibroblast, a neuronal cell, a glial cell, a lymphocyte (T cell or Bcell), or an insect cell; the polypeptide expression being monitored isXIAP, HIAP-1, HIAP-2, M-XIAP, M-HIAP-1, or M-HIAP-2 (i.e., human ormurine).

In a related aspect, the invention features methods of detectingcompounds that modulate apoptosis using the interaction trap technologyand IAP polypeptides, or fragments thereof, as a component of the bait.In preferred embodiments, the compound being tested as a modulator ofapoptosis is also a polypeptide.

In another aspect, the invention features a method for diagnosing a cellproliferation disease, or an increased likelihood of such a disease,using an IAP nucleic acid probe or antibody. Preferably, the disease isa cancer. Most preferably, the disease is selected from the groupconsisting of promyelocytic leukemia, a HeLa-type carcinoma, chronicmyelogenous leukemia (preferably using xiap or hiap-2 related probes),lymphoblastic leukemia (preferably using a xiap related probe),Burkitt's lymphoma (preferably using an hiap-1 related probe),colorectal adenocarcinoma, lung carcinoma, and melanoma (preferablyusing a xiap probe). Preferably, a diagnosis is indicated by a 2-foldincrease in is expression or activity, more preferably, at least a10-fold increase in expression or activity.

Skilled artisans will recognize that a mammalian IAP, or a fragmentthereof (as described herein), may serve as an active ingredient in atherapeutic composition. This composition, depending on the IAP orfragment included, may be used to modulate apoptosis and thereby treatany condition that is caused by a disturbance in apoptosis.

In addition, apoptosis may be induced in a cell by administering to thecell a negative regulator of the IAP-dependent anti-apoptotic pathway.The negative regulator may be, but is not limited to, an IAP polypeptidethat includes a ring zinc finger, and an LAP polypeptide that includes aring zinc finger and lacks at least one BIR domain. Alternatively,apoptosis may be induced in the cell by administering a gene encoding anIAP polypeptide, such as these two polypeptides. In yet another method,the negative regulator may be a purified antibody, or a fragmentthereof, that binds specifically to an IAP polypeptide. For example, theantibody may bind to an approximately 26 kDa cleavage product of an LAPpolypeptide that includes at least one BIR domain but lacks a ring zincfinger domain. The negative regulator may also be an IAP antisense mRNAmolecule.

As summarized above, an IAP nucleic acid, or an LAP polypeptide may beused to modulate apoptosis. Furthermore, an IAP nucleic acid, or an IAPpolypeptide, may be used in the manufacture of a medicament for themodulation of apoptosis.

By “IAP gene” is meant a gene encoding a polypeptide having at least oneBIR domain and a ring zinc finger domain which is capable of modulating(inhibiting or enhancing) apoptosis in a cell or tissue when provided byother intracellular or extracellular delivery methods. In preferredembodiments the IAP gene is a gene having about 50% or greaternucleotide sequence identity to at least one of the IAP amino acidencoding sequences of FIGS. 1-4 or portions thereof. Preferably, theregion of sequence over which identity is measured is a region encodingat least one BIR domain and a ring zinc finger domain. Mammalian IAPgenes include nucleotide sequences isolated from any mammalian source.Preferably, the mammal is a human.

The term “IAP gene” is meant to encompass any member of the family ofapoptosis inhibitory genes, which are characterized by their ability tomodulate apoptosis. An IAP gene may encode a polypeptide that has atleast 20%, preferably at least 30%, and most preferably at least 50%amino acid sequence identity with at least one of the conserved regionsof one of the IAP members described herein (i.e., either the BIR or ringzinc finger domains from the human or murine xiap, hiap-1 and hiap-2).Representative members of the IAP gene family include, withoutlimitation, the human and murine xiap, hiap-1, and hiap-2 genes.

By “IAP protein” or “IAP polypeptide” is meant a polypeptide, orfragment thereof, encoded by an IAP gene.

By “BIR domain” is meant a domain having the amino acid sequence of theconsensus sequence:Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val,wherein Xaa1 is any amino acid and Xaa2 is any amino acid or is absent(SEQ ID NO: 2). Preferably, the sequence is substantially identical toone of the BIR domain sequences provided herein for XIAP, HIAP-1, orHIAP-2.

By “ring zinc finger” or “RZF” is meant a domain having the amino acidsequence of the consensus sequence:Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,wherein Xaa1 is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val orIle (SEQ ID NO: 1).

Preferably, the sequence is substantially identical to the RZF domainsprovided herein for the human or murine XIAP, HIAP-1, or HIAP-2.

By “modulating apoptosis” or “altering apoptosis” is meant increasing ordecreasing the number of cells that would otherwise undergo apoptosis ina given cell population. Preferably, the cell population is selectedfrom a group including T cells, neuronal cells, fibroblasts, or anyother cell line known to undergo apoptosis in a laboratory setting(e.g., the baculovirus infected insect cells). It will be appreciatedthat the degree of modulation provided by an IAP or modulating compoundin a given assay will vary, but that one skilled in the art candetermine the statistically significant change in the level of apoptosiswhich identifies an IAP or a compound which modulates an IAP.

By “inhibiting apoptosis” is meant any decrease in the number of cellswhich undergo apoptosis relative to an untreated control. Preferably,the decrease is at least 25%, more preferably the decrease is 50%, andmost preferably the decrease is at least one-fold.

By “polypeptide” is meant any chain of more than two amino acids,regardless of post-translational modification such as glycosylation orphosphorylation.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 50%, preferably 85%, more preferably 90%, and mostpreferably 95% homology to a reference amino acid or nucleic acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids. For nucleic acids, the length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis softwarewith the default parameters specified therein (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine, valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

By “substantially pure polypeptide” is meant a polypeptide that has beenseparated from the components that naturally accompany it. Typically,the polypeptide is substantially pure when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the polypeptide is anIAP polypeptide that is at least 75%, more preferably at least 90%, andmost preferably at least 99%, by weight, pure. A substantially pure IAPpolypeptide may be obtained, for example, by extraction from a naturalsource (e.g. a fibroblast, neuronal cell, or lymphocyte) by expressionof a recombinant nucleic acid encoding an IAP polypeptide, or bychemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants which accompany it in itsnatural state. Thus, a protein which is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptidesinclude those derived from eukaryotic organisms but synthesized in E.coli or other prokaryotes. By “substantially pure DNA” is meant DNA thatis free of the genes which, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences. It also includes a recombinant DNA whichis part of a hybrid gene encoding additional polypeptide sequence.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding (as used herein) an IAP polypeptide.

By “transgene” is meant any piece of DNA which is inserted by artificeinto a cell, and becomes part of the genome of the organism whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism.

By “transgenic” is meant any cell which includes a DNA sequence which isinserted by artifice into a cell and becomes part of the genome of theorganism which develops from that cell. As used herein, the transgenicorganisms are generally transgenic mammalian (e.g., rodents such as ratsor mice) and the DNA (transgene) is inserted by artifice into thenuclear genome.

By “transformation” is meant any method for introducing foreignmolecules into a cell. Lipofection, calcium phosphate precipitation,retroviral delivery, electroporation, and biolistic transformation arejust a few of the teachings which may be used. For example, biolistictransformation is a method for introducing foreign molecules into a cellusing velocity driven microprojectiles such as tungsten or goldparticles. Such velocity-driven methods originate from pressure burstswhich include, but are not limited to, helium-driven, air-driven, andgunpowder-driven techniques. Biolistic transformation may be applied tothe transformation or transfection of a wide variety of cell types andintact tissues including, without limitation, intracellular organelles(e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi,algae, animal tissue, and cultured cells.

By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,an IAP polypeptide, a recombinant protein or a RNA molecule).

By “reporter gene” is meant a gene whose expression may be assayed; suchgenes include, without limitation, glucuronidase (GUS), luciferase,chloramphenicol transacetylase (CAT), and lacZ.

By “promoter” is meant minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific orinducible by external signals or agents; such elements may be located inthe 5′ or 3′ regions of the native gene.

By “operably linked” is meant that a gene and one or more regulatorysequences are connected in such a way as to permit gene expression whenthe appropriate molecules (e.g., transcriptional activator proteins arebound to the regulatory sequences).

By “conserved region” is meant any stretch of six or more contiguousamino acids exhibiting at least 30%, preferably 50%, and most preferably70% amino acid sequence identity between two or more of the IAP familymembers, (e.g., between human HIAP-1, HIAP-2, and XIAP). Examples ofpreferred conserved regions are shown (as boxed or designated sequences)in FIGS. 5-7 and Tables 1 and 2, and include, without limitation, BIRdomains and ring zinc finger domains.

By “detectably-labelled” is meant any means for marking and identifyingthe presence of a molecule, e.g., an oligonucleotide probe or primer, agene or fragment thereof, or a cDNA molecule. Methods fordetectably-labelling a molecule are well known in the art and include,without limitation, radioactive labelling (e.g., with an isotope such as³²P or ³⁵S) and nonradioactive labelling (e.g., chemiluminescentlabelling, e.g., fluorescein labelling).

By “antisense,” as used herein in reference to nucleic acids, is meant anucleic acid sequence, regardless of length, that is complementary tothe coding strand of a gene.

By “purified antibody” is meant antibody which is at least 60%, byweight, free from proteins and naturally occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, e.g., an IAP specific antibody. A purified antibodymay be obtained, for example, by affinity chromatography usingrecombinantly-produced protein or conserved motif peptides and standardtechniques.

By “specifically binds” is meant an antibody that recognizes and binds aprotein but that does not substantially recognize and bind othermolecules in a sample, e.g., a biological sample, that naturallyincludes protein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the human xiap cDNA sequence (SEQ ID NO: 3) and the XIAPpolypeptide sequence (SEQ ID NO: 4).

FIG. 2 is the human hiap-1 cDNA sequence (SEQ ID NO: 5) and the HIAP-1polypeptide sequence (SEQ ID NO: 6).

FIG. 3 is the human hiap-2 cDNA sequence (SEQ ID NO: 7) and the HIAP-2polypeptide sequence (SEQ ID NO: 8). The sequence absent in the hiap-2-Δvariant is boxed.

FIG. 4 is the murine xiap cDNA sequence (SEQ ID NO: 9) and encodedmurine XIAP polypeptide sequence (SEQ ID NO: 10).

FIG. 5 is the murine hiap-1 cDNA sequence (SEQ ID NO: 39) and theencoded murine HIAP-1 polypeptide sequence (SEQ ID NO: 40).

FIG. 6 is the murine hiap-2 cDNA sequence (SEQ ID NO: 41) and theencoded murine HIAP-2 polypeptide (SEQ ID NO: 42).

FIG. 7 is a representation of the alignment of the BIR domains of IAPproteins (SEQ ID NOs: 11 and 14-31).

FIG. 8 is a representation of the alignment of human IAP polypeptideswith diap, cp-iap, and the LAP consensus sequence (SEQ ID NOs: 4, 6, 8,10, 12, and 13).

FIG. 9 is a representation of the alignment of the ring zinc fingerdomains of IAP proteins (SEQ ID NOs: 32-38).

FIG. 10 is a photograph of a northern blot illustrating human hiap-1 andhiap-2 mRNA expression in human tissues.

FIG. 11 is a photograph of a northern blot illustrating human hiap-2mRNA expression in human tissues.

FIG. 12 is a photograph of a northern blot illustrating human xiap mRNAexpression in human tissues.

FIGS. 13A and 13B are photographs of agarose gels illustrating apoptoticDNA ladders and RT-PCR products using hiap-1 and hiap-2 specific probesin HIV-infected T cells.

FIG. 14A-14D are graphs depicting suppression of apoptosis by XIAP,HIAP-1, HIAP-2, Bcl-2, smn, and 6-myc.

FIGS. 15A and 15B are bar graphs depicting the percentage of viable CHOcells following transient transfection with the cDNA constructs shownand subsequent serum withdrawal.

FIGS. 16A and 16B are bar graphs depicting the percentage of viable CHOcells following transient transfection with the cDNA constructs shownand subsequent exposure to menadione (FIG. 16A=10 μM menadione; FIG.16B=20 μM menadione).

FIG. 17 is a photograph of an agarose gel containing cDNA fragments thatwere amplified, with hiap-1-specific primers, from RNA obtained fromRaji, Ramos, EB-3, and Jiyoye cells, and from normal placenta.

FIG. 18 is a photograph of a western blot containing protein extractedfrom Jurkat and astrocytoma cells stained with an anti-XIAP antibody.The position and size of a series of marker proteins is indicated.

FIG. 19 is a photograph of a western blot containing protein extractedfrom Jurkat cells following treatment as described in Example XII. Theblot was stained with a rabbit polyclonal anti-XIAP antibody. Lane 1,negative control; lane 2, anti-Fas antibody; lane 3, anti-Fas antibodyand cycloheximide; lane 4, TNF-α; lane 5, TNF-α and cycloheximide.

FIG. 20 is a photograph of a western blot containing protein extractedfrom HeLa cells following exposure to anti-Fas antibodies. The blot wasstained with a rabbit polyclonal anti-XIAP antibody. Lane 1, negativecontrol; lane 2, cycloheximide; lane 3, anti-Fas antibody; lane 4,anti-Fas antibody and cycloheximide; lane 5, TNF-α; lane 6, TNF-α andcycloheximide.

FIGS. 21A and 21B are photographs of western blots stained with rabbitpolyclonal anti-XIAP antibody. Protein was extracted from HeLa cells(FIG. 21A) and Jurkat cells (FIG. 21B) immediately, 1, 2, 3, 5, 10, and22 hours after exposure to anti-Fas antibody.

FIGS. 22A and 22B are photographs of western blots stained with ananti-CPP32 antibody (FIG. 22A) or a rabbit polyclonal anti-XIAP antibody(FIG. 22B). Protein was extracted from Jurkat cells immediately, 3hours, or 7 hours after exposure to an anti-Fas antibody. In addition tototal protein, cytoplasmic and nuclear extracts are shown.

FIG. 23 is a photograph of a polyacrylamide gel followingelectrophoresis of the products of an in vitro XIAP cleavage assay.

DETAILED DESCRIPTION

I. IAP Genes and Polypeptides

A new class of mammalian proteins that modulate apoptosis (IAPs) and thegenes that encode these proteins have been discovered. The IAP proteinsare characterized by the presence of a ring zinc finger domain (RZF;FIG. 9) and at least one BIR domain, as defined by the boxed consensussequences shown in FIGS. 7 and 8, and by the sequence domains listed inTables 1 and 2. As examples of novel IAP genes and proteins, the cDNAsequences and amino acid sequences for human IAPs (HIAP-1, HIAP-2, andXIAP) and a new murine inhibitor of apoptosis, XIAP, are provided.Additional members of the mammalian IAP family (including homologs fromother species and mutant sequences) may be isolated using standardcloning techniques and the conserved amino acid sequences, primers, andprobes provided herein and known in the art. Furthermore, IAPs includethose proteins lacking the ring zinc finger, as further described below.TABLE 1 NUCLEOTIDE POSITION OF CONSERVED DOMAINS* Ring Zinc BIR-1 BIR-2BIR-3 Finger h-xiap 109-312 520-723 826-1023 1348-1485 m-xiap 202-405613-816 916-1113 1438-1575 h-hiap-1 273-476 693-893 951-1154 1824-1961m-hiap-1 251-453 670-870 928-1131 1795-1932 h-hiap-2 373-576 787-9871042-1245  1915-2052 m-hiap-2 215-418 608-808 863-1066 1763-1876*Positions indicated correspond to those shown in FIGS. 1-4.

TABLE 2 AMINO ACID POSITION OF CONSERVED DOMAINS* Ring Zinc BIR-1 BIR-2BIR-3 Finger h-XAIP 26-93 163-230 265-330 439-484 m-XIAP 26-93 163-230264-329 438-483 h-HIAP1 29-96 169-235 255-322 546-591 m-HIAP1 29-96169-235 255-322 544-589 h-HIAP2  46-113 184-250 269-336 560-605 m-HIAP225-92 156-222 241-308 541-578*Positions indicated correspond to those shown in FIGS. 1-4.

Recognition of the mammalian LAP family has provided an emergent patternof protein structure. Recognition of this pattern allows proteins havinga known, homologous sequence but unknown function to be classified asputative inhibitors of apoptosis. A Drosophila gene, now termed diap,was classified in this way (for sequence information see GenbankAccession Number M96581 and FIG. 6). The conservation of these proteinsacross species indicates that the apoptosis signalling pathway has beenconserved throughout evolution.

The IAP proteins may be used to inhibit the apoptosis that occurs aspart of numerous disease processes or disorders. For example, IAPpolypeptides or nucleic acid encoding LAP polypeptides may beadministered for the treatment or prevention of apoptosis that occurs asa part of AIDS, neurodegenerative diseases, ischemic injury,toxin-induced liver disease and myelodysplastic syndromes. Nucleic acidencoding the IAP polypeptide may also be provided to inhibit apoptosis.

II. Cloning of IAP Genes

A. Human Xiap

The search for human genes involved in apoptosis resulted in theidentification of an X-linked sequence tag site (STS) in the GenBankdatabase, which demonstrated strong homology with the conserved RZFdomain of CpIAP and OpIAP, the two baculovirus genes known to inhibitapoptosis (Clem et al., Mol. Cell Biol. 14:5212, 1994; Birnbaum et al.,J. Virol. 68:2521, 1994). Screening a human fetal brain ZapII cDNAlibrary (Stratagene, La Jolla, Calif.) with this STS resulted in theidentification and cloning of xiap (for X-linked Inhibitor of ApoptosisProtein gene). The human gene has a 1.5 kb coding sequence that includesthree BIR domains (Crook et al., J. Virol. 67:2168, 1993; Clem et al.,Science 254:1388, 1991; Birnbaum et al., J. Virol. 68:2521, 1994) and azinc finger. Northern blot analysis with xiap revealed message greaterthan 7 kb, which is expressed in various tissues, particularly liver andkidney (FIG. 12). The large size of the transcript reflects large 5′ and3′ untranslated regions.

B. Human Hiap-1 and Hiap-2

The hiap-1 and hiap-2 genes were cloned by screening a human liverlibrary (Stratagene Inc., LaJolla, Calif.) with a probe including theentire xiap coding region at low stringency (the final wash wasperformed at 40° C. with 2×SSC, 10% SDS; FIGS. 2 and 3). The hiap-1 andhiap-2 genes were also detected independently using a probe derived froman expressed sequence tag (EST; GenBank Accession No. T96284), whichincludes a portion of a BIR domain. The EST sequence was originallyisolated by the polymerase chain reaction; a cDNA library was used as atemplate and amplified with EST-specific primers. The DNA amplifiedprobe was then used to screen the human liver cDNA library forfull-length hiap coding sequences. A third DNA was subsequently detectedthat includes the hiap-2 sequence but that appears to lack one exon,presumably due to alternative mRNA splicing (see boxed region in FIG.3). The expression of hiap-1 and hiap-2 in human tissues as assayed bynorthern blot analysis is shown in FIGS. 8 and 9.

C. M-Xiap

Fourteen cDNA and two genomic clones were identified by screening amouse embryo λgt11 cDNA library (Clontech, Palo Alto, Calif.) and amouse FIX II genomic library with a xiap cDNA probe, respectively. AcDNA contig spanning 8.0 kb was constructed using 12 overlapping mouseclones. Sequence analysis revealed a coding sequence of approximately1.5 kb. The mouse gene, m-xiap, encodes a polypeptide with strikinghomology to human XIAP at and around the initiation methionine, the stopcodon, the three BIR domains, and the RZF domain. As with the humangene, the mouse homologue contains large 5′ and 3′ UTRs, which couldproduce a transcript as large as 7-8 kb.

Analysis of the sequence and restriction map of m-xiap further delineatethe structure and genomic organization of m-xiap. Southern blot analysisand inverse PCR techniques (Groden et al., Cell 66:589, 1991) can beemployed to map exons and define exon-intron boundaries.

Antisera can be raised against a M-XIAP fusion protein that was obtainedfrom, for example, E. coli using a bacterial expression system. Theresulting antisera can be used along with northern blot analysis toanalyze the spatial and temporal expression of m-xiap in the mouse.

D. M-Hiap-1 and M-Hiap-2

The murine homologs of hiap-1 and hiap-2 were cloned and sequenced inthe same general manner as m-xiap using the human hiap-1 and hiap-2sequences as probes. Cloning of m-hiap-1 and m-hiap-2 furtherdemonstrate that homologs from different species may be isolated usingthe techniques provided herein and those generally known to artisansskilled in molecular biology.

III. Identification of Additional IAP Genes

Standard techniques, such as the polymerase chain reaction (PCR) and DNAhybridization, may be used to clone additional human IAP genes and theirhomologues in other species. Southern blots of human genomic DNAhybridized at low stringency with probes specific for xiap, hiap-1 andhiap-2 reveal bands that correspond to other known human IAP sequencesas well as additional bands that do not correspond to known IAPsequences. Thus, additional IAP sequences may be readily identifiedusing low stringency hybridization. Examples of murine and human xiap,hiap-1, and hiap-2 specific primers, which may be used to cloneadditional genes by RT-PCR, are shown in Table 5.

IV. Characterization of IAP Activity and Intracellular LocalizationStudies

The ability of putative IAPs to modulate apoptosis can be defined in invitro systems in which alterations of apoptosis can be detected.Mammalian expression constructs carrying IAP cDNAs, which are eitherfull-length or truncated, can be introduced into cell lines such as CHO,NIH 3T3, HL60, Rat-1, or Jurkat cells. In addition, Sf21 insect cellsmay be used, in which case the IAP gene is preferentially expressedusing an insect heat shock promotor. Following transfection, apoptosiscan be induced by standard methods, which include serum withdrawal, orapplication of staurosporine, menadione (which induces apoptosis viafree radial formation), or anti-Fas antibodies. As a control, cells arecultured under the same conditions as those induced to undergoapoptosis, but either not transfected, or transfected with a vector thatlacks an IAP insert. The ability of each IAP construct to inhibitapoptosis upon expression can be quantified by calculating the survivalindex of the cells, i.e., the ratio of surviving transfected cells tosurviving control cells. These experiments can confirm the presence ofapoptosis inhibiting activity and, as discussed below, can also be usedto determine the functional region(s) of an IAP. These assays may alsobe performed in combination with the application of additional compoundsin order to identify compounds that modulate apoptosis via IAPexpression.

A. Cell Survival Following Transfection with Full-Length IAP Constructsand Induction of Apoptosis

Specific examples of the results obtained by performing variousapoptosis suppression assays are shown in FIGS. 14A to 14D. For example,CHO cell survival following transfection with one of six constructs andsubsequent serum withdrawal is shown in FIG. 14A. The cells weretransfected using Lipofectace™ with 2 μg of one of the followingrecombinant plasmids: pcDNA3-6myc-xiap (xiap), pcDNA3-6myc-hiap-1(hiap-1), pcDNA3-6myc-hiap-2 (hiap-2), pcDNA3-bcl-2 (bcl-2),pcDNA3-HA-smn (smn), and pcDNA3-6myc (6-myc). Oligonucleotide primerswere synthesized to allow PCR amplification and cloning of the xiap,hiap-1, and hiap-2 ORFs in pcDNA3 (Invitrogen). Each construct wasmodified to incorporate a synthetic myc tag encoding six repeats of thepeptide sequence MEQKLISEEDL (SEQ ID NO: 43), thus allowing detection ofmyc-IAP fusion proteins via monoclonal anti-myc antiserum (Egan et al.,Nature 363:45, 1993). Triplicate samples of cell lines in 24-well disheswere washed 5 times with serum free media and maintained in serum freeconditions during the course of the experiment. Cells that excludedtrypan blue, and that were therefore viable, were counted with ahemocytometer immediately, 24 hours, 48 hours, and 72 hours after serumwithdrawal. Survival was calculated as a percentage of the initialnumber of viable cells. In this experiment, as well as those presentedin FIGS. 14B and 14D, the percentage of viable cells shown representsthe average of three separate experiments performed in triplicate,standard deviation.

The survival of CHO cells following transfection (with each one of thesix constructs described above) and exposure to menadione is shown inFIG. 14B. The cells were plated in 24-well dishes, allowed to growovernight, and then exposed to 20 μM menadione (Sigma Chemical Co., St.Louis, Mo.) for 1.5 hours. Triplicate samples were harvested at the timeof exposure to menadione and 24 hours afterward, and survival wasassessed by trypan blue exclusion.

The survival of Rat-1 cells following transfection (with each one of thesix constructs described above) and exposure to staurosporine is shownin FIG. 14C. Rat-1 cells were transfected and then selected in mediumcontaining 800 μg/ml G418 for two weeks. The cell line was assessed forresistance to staurosporine-induced apoptosis (1 μM) for 5 hours. Viablecells were counted 24 hours after exposure to staurosporine by trypanblue exclusion. The percentage of viable cells shown represents theaverage of two experiments, ±standard deviation.

The Rat-1 cell line was also used to test the resistance of these cellsto menadione (FIG. 14D) following transfection with each of the sixconstructs described above. The cells were exposed to 10 μM menadionefor 1.5 hours, and the number of viable cells was counted 18 hourslater.

B. Comparison of Cell Survival Following Transfection with Full-Lengthvs. Partial IAP Constructs

In order to investigate the mechanism whereby human IAPs, includingXIAP, HIAP-1, and HIAP-2, afford protection against cell death,expression vectors were constructed that contained either: (1)full-length IAP cDNA (as described above), (2) a portion of an IAP genethat encodes the BIR domains, but not the RZF, or (3) a portion of anIAP gene that encodes the RZF, but not the BIR domains. Human and murinexiap or m-xiap cDNAs were tested by transient or stable expression inHeLa, Jurkat, and CHO cell lines. Following transfection, apoptosis wasinduced by serum withdrawal, application of menadione, or application ofan anti-Fas antibody. Cell death was then assessed, as described above,by trypan blue exclusion. As a control for transfection efficiency, thecells were co-transfected with a β-gal expression construct. Typically,approximately 20% of the cells were successfully transfected.

When CHO cells were transiently transfected, constructs containingfull-length xiap or m-xiap cDNAs conferred modest protection againstcell death (FIG. 15A). In contrast, the survival of CHO cellstransfected with constructs encoding only the BIR domains (i.e., lackingthe RZF domain; see FIG. 15A) was markedly enhanced 72 hours after serumdeprivation. Furthermore, a large percentage of cells expressing the BIRdomains were still viable after 96 hours, at which time no viable cellsremained in the control, i.e. non-transfected, cell cultures (see “CHO”in FIG. 15A), and less than 5% of the cells transfected with the vectoronly, i.e., lacking a cDNA insert, remained viable (see “pcDNA3” in FIG.15A). Deletion of any of the BIR domains results in the complete loss ofis apoptotic suppression, which is reflected by a decrease in thepercentage of surviving CHO cells to control levels within 72 hours ofserum withdrawal (FIG. 15B; see “xiapΔ1” (which encodes amino acids89-497 of XIAP (SEQ ID NO.:4)), “xiapΔ2” (which encodes amino acids246497 of XIAP (SEQ ID NO.:4)), and “xiapΔ3” (which encodes amino acids342497 of XIAP (SEQ ID NO.:4)) at 72 hours).

Stable pools of transfected CHO cells, which were maintained for severalmonths under G418 selection, were induced to undergo apoptosis byexposure to 10 μM menadione for 2 hours. Among the CHO cells tested werethose that were stably transfected with: (1) full-length m-xiap cDNA(miap), (2) full-length xiap cDNA (xiap), (3) full-length bcl-2 cDNA(Bcl-2), (4) cDNA encoding the three BIR domains (but not the RZF) ofM-XIAP (BIR), and (5) cDNA encoding the RZF (but not BIR domains) ofM-XIAP (RZF). Cells that were non-transfected (CHO) or transfected withthe vector only (pcDNA3), served as controls for this experiment.Following exposure to 10 μM menadione, the transfected cells were washedwith phosphate buffered saline (PBS) and cultured for an additional 24hours in menadione-free medium. Cell death was assessed, as describedabove, by trypan blue exclusion. Less than 10% of the non-transfected orvector-only transfected cells remained viable at the end of the 24 hoursurvival period. Cells expressing the RZF did not fare significantlybetter. However, expression of full-length m-xiap, xiap, or bcl-2, andexpression of the BIR domains, enhanced cell survival (FIG. 16A). Whenthe concentration of menadione was increased from 10 μM to 20 μM (withall other conditions of the experiment being the same as when 10 μMmenadione was applied), the percentage of viable CHO cells thatexpressed the BIR domain cDNA construct was higher than the percentageof viable cells that expressed either full-length m-xiap or bcl-2 (FIG.16B).

C. Analysis of the Subcellular Location of Expressed RZF and BIR Domains

The assays of cell death described above indicate that the RZF may actas a negative regulator of the anti-apoptotic function of IAPs. One wayin which the RZF, and possibly other IAP domains, may exert theirregulatory influence is by altering the expression of genes, whoseproducts function in the apoptotic pathway.

In order to determine whether the subcellular locations of expressed RZFand BIR domains are consistent with roles as nuclear regulatory factors,COS cells were transiently transfected with the following fourconstructs, and the expressed polypeptide was localized byimmunofluorescence microscopy: (1) pcDNA3-6myc-xiap, which encodes all497 amino acids of SEQ ID NO: 4, (2) pcDNA3-6myc-m-xiap, which encodesall 497 amino acids of mouse xiap (SEQ ID NO: 10), (3)pcDNA3-6myc-mxiap-BIR, which encodes amino acids 1 to 341 of m-xiap (SEQID NO: 10), and (4) pcDNA3-6myc-mxiap-RZF, which encodes amino acids342-497 of m-xiap (SEQ ID NO: 10). The cells were grown on multi-welltissue culture slides for 12 hours, and then fixed and permeabilizedwith methanol. The constructs used (here and in the cell death assays)were tagged with a human Myc epitope tag at the N-terminus. Therefore, amonoclonal anti-Myc antibody and a secondary goat anti-mouse antibody,which was conjugated to FITC, could be used to localize the expressedproducts in transiently transfected COS cells. Full-length XIAP and MIAPwere located in the cytoplasm, with accentuated expression in theperi-nuclear zone. The same pattern of localization was observed whenthe cells expressed a construct encoding the RZF domain (but not the BIRdomains). However, cells expressing the BIR domains (without the RZF)exhibited, primarily, nuclear staining. The protein expressed by the BIRdomain construct appeared to be in various stages of transfer to thenucleus.

These observations are consistent with the fact that, as describedbelow, XIAP is cleaved within T cells that are treated with anti-Fasantibodies (which are potent inducers of apoptosis), and its N-terminaldomain is translocated to the nucleus.

D. Examples of Additional Apoptosis Assays

Specific examples of apoptosis assays are also provided in the followingreferences. Assays for apoptosis in lymphocytes are disclosed by: Li etal., Science 268:429, 1995; Gibellini et al., Br. J. Haematol. 89:24,1995; Martin et al., J. Immunol. 152:330, 1994; Terai et al., J. Clin.Invest. 87:1710, 1991; Dhein et al., Nature 373:438, 1995; Katsikis etal., J. Exp. Med. 1815:2029, 1995; Westendorp et al., Nature 375:497,1995; DeRossi et al., Virology 198:234, 1994.

Assays for apoptosis in fibroblasts are disclosed by: Vossbeck et al.,Int. J. Cancer 61:92, 1995; Goruppi et al., Oncogene 9:1537, 1994;Fernandez et al., Oncogene 9:2009, 1994; Harrington et al., EMBO J.,13:3286, 1994; Itoh et al., J. Biol. Chem. 268:10932, 1993.

Assays for apoptosis in neuronal cells are disclosed by: Melino et al.,Ann. Neurol. 36:864, 1994; Sato et al., J. Neurobiol. 25:1227, 1994;Ferrari et al., J. Neurosci. 1516:2857, 1995; Talley et al., Mol. CellBiol. 1585:2359, 1995; Talley et al., Mol. Cell. Biol. 15:2359, 1995;Walkinshaw et al., J. Clin. Invest. 95:2458, 1995.

Assays for apoptosis in insect cells are disclosed by: Clem et al.,Science 254:1388, 1991; Crook et al., J. Virol. 67:2168, 1993; Rabizadehet al., J. Neurochem. 61:2318, 1993; Birnbaum et al., J. Virol. 68:2521,1994; Clem et al., Mol. Cell. Biol. 14:5212, 1994.

V. Construction of a Transgenic Animal

Characterization of IAP genes provides information that is necessary foran IAP knockout animal model to be developed by homologousrecombination. Preferably, the model is a mammalian animal, mostpreferably a mouse. Similarly, an animal model of IAP overproduction maybe generated by integrating one or more LAP sequences into the genome,according to standard transgenic techniques.

A replacement-type targeting vector, which would be used to create aknockout model, can be constructed using an isogenic genomic clone, forexample, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla,Calif.). The targeting vector is introduced into a suitably-derived lineof embryonic stem (ES) cells by electroporation to generate ES celllines that carry a profoundly truncated form of an IAP. To generatechimeric founder mice, the targeted cell lines are injected into a mouseblastula stage embryo. Heterozygous offspring are interbred tohomozygosity. Knockout mice would provide the means, in vivo, to screenfor therapeutic compounds that modulate apoptosis via an IAP-dependentpathway.

VI. IAP Protein Expression

IAP genes may be expressed in both prokaryotic and eukaryotic celltypes. If an LAP modulates apoptosis by exacerbating it, it may bedesirable to express that protein under control of an induciblepromotor.

In general, IAPs according to the invention may be produced bytransforming a suitable host cell with all or part of an LAP-encodingcDNA fragment that has been placed into a suitable expression vector.

Those skilled in the art of molecular biology will understand that awide variety of expression systems may be used to produce therecombinant protein. The precise host cell used is not critical to theinvention. The IAP protein may be produced in a prokaryotic host (e.g.,E. coli) or in a eukaryotic host (e.g., S. cerevisiae, insect cells suchas Sf21 cells, or mammalian cells such as COS-1, NIH 3T3, or HeLacells). These cells are publicly available, for example, from theAmerican Type Culture Collection (ATCC), Rockville, Md.; see alsoAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1994. The method of transduction and the choice ofexpression vehicle will depend on the host system selected.Transformation and transfection methods are described, e.g., in Ausubelet al. (supra), and expression vehicles may be chosen from thoseprovided, e.g. in Cloning Vectors: A Laboratory Manual (P. H. Pouwels etal., 1985, Supp. 1987).

A preferred expression system is the baculovirus system using, forexample, the vector pBacPAK9, which is available from Clontech (PaloAlto, Calif.). If desired, this system may be used in conjunction withother protein expression techniques, for example, the myc tag approachdescribed by Evan et al. (Mol. Cell Biol. 5:3610, 1985).

Alternatively, an IAP may be produced by a stably-transfected mammaliancell line. A number of vectors suitable for stable transfection ofmammalian cells are available to the public, (e.g., see Pouwels et al.,supra), as are methods for constructing such cell lines (e.g., seeAusubel et al., supra). In one example, cDNA encoding an IAP is clonedinto an expression vector that includes the dihydrofolate reductase(DHFR) gene. Integration of the plasmid and, therefore, integration ofthe IAP-encoding gene into the host cell chromosome is selected for byinclusion of 0.01-300 μM methotrexate in the cell culture medium (asdescribed in Ausubel et al., supra). This dominant selection can beaccomplished in most cell types. Recombinant protein expression can beincreased by DHFR-mediated amplification of the transfected gene.

Methods for selecting cell lines bearing gene amplifications aredescribed in Ausubel et al. (supra). These methods generally involveextended culture in medium containing gradually increasing levels ofmethotrexate. The most commonly used DHFR-containing expression vectorsare pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., supra).The host cells described above or, preferably, a DHFR-deficient CHO cellline (e.g., CHO DHFR⁻ cells, ATCC Accession No. CRL 9096) are amongthose most preferred for DHFR selection of a stably-transfected cellline or DHFR-mediated gene amplification.

Once the recombinant protein is expressed, it is isolated by, forexample, affinity chromatography. In one example, an anti-IAP antibody,which may be produced by the methods described herein, can be attachedto a column and used to isolate the IAP protein. Lysis and fractionationof IAP-harboring cells prior to affinity chromatography may be performedby standard methods (see e.g., Ausubel et al., supra). Once isolated,the recombinant protein can, if desired, be purified further by e.g., byhigh performance liquid chromatography (HPLC; e.g., see Fisher,Laboratory Techniques In Biochemistry And Molecular Biology, Work andBurdon, Eds., Elsevier, 1980).

Polypeptides of the invention, particularly short IAP fragments, canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co.,Rockford, Ill.). These general techniques of polypeptide expression andpurification can also be used to produce and isolate useful IAPfragments or analogs, as described herein.

VII. Anti-IAP Antibodies

In order to generate IAP-specific antibodies, an IAP coding sequence(i.e., amino acids 180-276) can be expressed as a C-terminal fusion withglutathione S-transferase (GST; Smith et al., Gene 67:31, 1988). Thefusion protein can be purified on glutathione-Sepharose beads, elutedwith glutathione, and cleaved with thrombin (at the engineered cleavagesite), and purified to the degree required to successfully immunizerabbits. Primary immunizations can be carried out with Freund's completeadjuvant and subsequent immunizations performed with Freund's incompleteadjuvant. Antibody titres are monitored by western blot andimmunoprecipitation analyses using the thrombin-cleaved IAP fragment ofthe GST-IAP fusion protein. Immune sera are affinity purified usingCNBr-Sepharose-coupled IAP protein. Antiserum specificity is determinedusing a panel of unrelated GST proteins (including GSTp53, Rb, HPV-16E6, and E6-AP) and GST-trypsin (which was generated by PCR using knownsequences).

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique hydrophilic regions of IAP may begenerated and coupled to keyhole limpet hemocyanin (KLH) through anintroduced C-terminal lysine. Antiserum to each of these peptides issimilarly affinity purified on peptides conjugated to BSA, andspecificity is tested by ELISA and western blotting using peptideconjugates, and by western blotting and immunoprecipitation using IAPexpressed as a GST fusion protein.

Alternatively, monoclonal antibodies may be prepared using the IAPproteins described above and standard hybridoma technology (see, e.g.,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981; Ausubel et al., supra). Once produced, monoclonal antibodiesare also tested for specific IAP recognition by western blot orimmunoprecipitation analysis (by the methods described in Ausubel etal., supra).

Antibodies that specifically recognize IAPs or fragments of IAPs, suchas those described herein containing one or more BIR domains (but not aring zinc finger domain), or that contain a ring zinc finger domain (butnot a BIR domain) are considered useful in the invention. They may, forexample, be used in an immunoassay to monitor IAP expression levels orto determine the subcellular location of an LAP or IAP fragment producedby a mammal. Antibodies that inhibit the 26 kDa IAP cleavage productdescribed herein (which contains at least one BIR domain) may beespecially useful in inducing apoptosis in cells undergoing undesirableproliferation.

Preferably, antibodies of the invention are produced using IAP sequencethat does not reside within highly conserved regions, and that appearslikely to be antigenic, as analyzed by criteria such as those providedby the Peptide structure program (Genetics Computer Group SequenceAnalysis Package, Program Manual for the GCG Package, Version 7, 1991)using the algorithm of Jameson and Wolf (CABIOS 4:181, 1988).Specifically, these regions, which are found between BIR1 and BIR2 ofall IAPs, are: amino acid 99 to amino acid 170 of HIAP-1, amino acid 123to amino acid 184 of HIAP-2, and amino acid 116 to amino acid 133 ofeither XIAP or M-XIAP. These fragments can be generated by standardtechniques, e.g., by PCR, and cloned into the pGEX expression vector(Ausubel et al., supra). Fusion proteins are expressed in E. coli andpurified using a glutathione agarose affinity matrix as described inAusubel et al. (supra). In order to minimize the potential for obtainingantisera that is non-specific, or exhibits low-affinity binding to IAP,two or three fusions are generated for each protein, and each fusion isinjected into at least two rabbits. Antisera are raised by injections inseries, preferably including at least three booster injections.

VII. Identification of Molecules that Modulate IAP Protein Expression

Isolation of IAP cDNAs also facilitates the identification of moleculesthat increase or decrease IAP expression. In one approach, candidatemolecules are added, in varying concentration, to the culture medium ofcells expressing IAP mRNA. IAP expression is then measured, for example,by northern blot analysis (Ausubel et al., supra) using an IAP cDNA, orcDNA fragment, as a hybridization probe (see also Table 5). The level ofIAP expression in the presence of the candidate molecule is compared tothe level of IAP expression in the absence of the candidate molecule,all other factors (e.g. cell type and culture conditions) being equal.

The effect of candidate molecules on IAP-mediated apoptosis may,instead, be measured at the level of translation by using the generalapproach described above with standard protein detection techniques,such as western blotting or immunoprecipitation with an LAP-specificantibody (for example, the IAP antibody described herein).

Compounds that modulate the level of LAP may be purified, orsubstantially purified, or may be one component of a mixture ofcompounds such as an extract or supernatant obtained from cells (Ausubelet al., supra). In an assay of a mixture of compounds, LAP expression istested against progressively smaller subsets of the compound pool (e.g.,produced by standard purification techniques such as HPLC or FPLC) untila single compound or minimal number of effective compounds isdemonstrated to modulate IAP expression.

Compounds may also be screened for their ability to modulate LAPapoptosis inhibiting activity. In this approach, the degree of apoptosisin the presence of a candidate compound is compared to the degree ofapoptosis in its absence, under equivalent conditions. Again, the screenmay begin with a pool of candidate compounds, from which one or moreuseful modulator compounds are isolated in a step-wise fashion.Apoptosis activity may be measured by any standard assay, for example,those described herein.

Another method for detecting compounds that modulate the activity ofIAPs is to screen for compounds that interact physically with a givenIAP polypeptide. These compounds may be detected by adapting interactiontrap expression systems known in the art. These systems detect proteininteractions using a transcriptional activation assay and are generallydescribed by Gyuris et al. (Cell 75:791, 1993) and Field et al. (Nature340:245, 1989), and are commercially available from Clontech (Palo Alto,Calif.). In addition, PCT Publication WO 95/28497 describes aninteraction trap assay in which proteins involved in apoptosis, byvirtue of their interaction with Bcl-2, are detected. A similar methodmay be used to identify proteins and other compounds that interact withIAPs.

Compounds or molecules that function as modulators of IAP-mediated celldeath may include peptide and non-peptide molecules such as thosepresent in cell extracts, mammalian serum, or growth medium in whichmammalian cells have been cultured.

A molecule that promotes an increase in IAP expression or LAP activityis considered particularly useful in the invention; such a molecule maybe used, for example, as a therapeutic to increase cellular levels ofIAP and thereby exploit the ability of IAP polypeptides to inhibitapoptosis.

A molecule that decreases IAP activity (e.g., by decreasing LAP geneexpression or polypeptide activity) may be used to decrease cellularproliferation. This would be advantageous in the treatment of neoplasms(see Table 3, below), or other cell proliferative diseases. TABLE 3NORTHERN BLOT IAP RNA LEVELS IN CANCER CELLS* xiap hiap1 hiap2Promyelocytic Leukemia HL-60 + + + Hela S-3 + + + Chronic MyelogenousLeukemia K-562 +++ + +++ Lymphoblastic Leukemia MOLT-4 +++ + + Burkitt'sLymphoma Raji + +(×10) + Colorectal Adenocarcinoma SW-480 +++ +++ +++Lung Carcinoma A-549 + + + Melanoma G-361 +++ + +*Levels are indicated by a (+) and are the approximate increase in RNAlevels relative to northern blots of RNA from non-cancerous control celllines. A single plus indicates an estimated increase of at least 1-fold

Molecules that are found, by the methods described above, to effectivelymodulate IAP gene expression or polypeptide activity may be testedfurther in animal models. If they continue to function successfully inan in vivo setting, they may be used as therapeutics to either inhibitor enhance apoptosis, as appropriate.

IX. IAP Therapy

The level of IAP gene expression correlates with the level of apoptosis.Thus, IAP genes also find use in anti-apoptosis gene therapy. Inparticular, a functional IAP gene may be used to sustain neuronal cellsthat undergo apoptosis in the course of a neurodegenerative disease,lymphocytes (i.e., T cells and B cells), or cells that have been injuredby ischemia.

Retroviral vectors, adenoviral vectors, adeno-associated viral vectors,or other viral vectors with the appropriate tropism for cells likely tobe involved in apoptosis (for example, epithelial cells) may be used asa gene transfer delivery system for a therapeutic IAP gene construct.Numerous vectors useful for this purpose are generally known (Miller,Human Gene Therapy 15, 1990; Friedman, Science 244:1275, 1989; Eglitisand Anderson, Biotechniques 6:608, 1988; Tolstoshev and Anderson, Curr.Opin. Biotechnol. 1:55, 1990; Sharp, Lancet 337:1277, 1991; Cornetta etal., Nucleic Acid Research and Molecular Biology 36:311, 1987; Anderson,Science 226:401, 1984; Moen, Blood Cells 17:407, 1991; Miller et al.,Biotechniques 7:980, 1989; La Salle et al., Science 259:988, 1993;Johnson, Chest 107:77S, 1995). Retroviral vectors are particularly welldeveloped and have been used in clinical settings (Rosenberg et al., N.Engl. J. Med. 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).Non-viral approaches may also be employed for the introduction oftherapeutic DNA into cells otherwise predicted to undergo apoptosis. Forexample, IAP may be introduced into a neuron or a T cell by lipofection(Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al.,Neurosci. Lett. 117:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278,1989; Staubinger et al., Meth. Enzymol. 101:512, 1983),asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem.263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989); or, lesspreferably, microinjection under surgical conditions (Wolff et al.,Science 247:1465, 1990).

For any of the methods of application described above, the therapeuticIAP DNA construct is preferably applied to the site of the predictedapoptosis event (for example, by injection). However, it may also beapplied to tissue in the vicinity of the predicted apoptosis event or toa blood vessel supplying the cells predicted to undergo apoptosis.

In the constructs described, IAP cDNA expression can be directed fromany suitable promoter (e.g., the human cytomegalovirus (CMV), simianvirus (SV40), or metallothionein promoters), and regulated by anyappropriate mammalian regulatory element. For example, if desired,enhancers known to preferentially direct gene expression in neuralcells, T cells, or B cells may be used to direct IAP expression. Theenhancers used could include, without limitation, those that arecharacterized as tissue- or cell-specific in their expression.Alternatively, if an IAP genomic clone is used as a therapeuticconstruct (for example, following its isolation by hybridization withthe IAP cDNA described above), regulation may be mediated by the cognateregulatory sequences or, if desired, by regulatory sequences derivedfrom a heterologous source, including any of the promoters or regulatoryelements described above.

Less preferably, IAP gene therapy is accomplished by directadministration of the IAP mRNA or antisense IAP mRNA to a cell that isexpected to undergo apoptosis. The mRNA may be produced and isolated byany standard technique, but is most readily produced by in vitrotranscription using an IAP is cDNA under the control of a highefficiency promoter (e.g., the T7 promoter). Administration of IAP mRNAto malignant cells can be carried out by any of the methods for directnucleic acid administration described above.

Ideally, the production of IAP protein by any gene therapy approach willresult in cellular levels of IAP that are at least equivalent to thenormal, cellular level of IAP in an unaffected cell. Treatment by anyIAP-mediated gene therapy approach may be combined with more traditionaltherapies.

Another therapeutic approach within the invention involvesadministration of recombinant IAP protein, either directly to the siteof a predicted apoptosis event (for example, by injection) orsystemically (for example, by any conventional recombinant proteinadministration technique). The dosage of IAP depends on a number offactors, including the size and health of the individual patient, but,generally, between 0.1 mg and 100 mg inclusive are administered per dayto an adult in any pharmaceutically acceptable formulation.

X. Administration of IAP Polypeptides, IAP Genes, or Modulators of LAPSynthesis or Function

An IAP protein, gene, or modulator may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer IAP topatients suffering from a disease that is caused by excessive apoptosis.Administration may begin before the patient is symptomatic. Anyappropriate route of administration may be employed, for example,administration may be parenteral, intravenous, intraarterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, aerosol, or oral administration.Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for LAP modulatory compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

If desired, treatment with an LAP protein, gene, or modulatory compoundmay be combined with more traditional therapies for the disease such assurgery, steroid therapy, or chemotherapy for autoimmune disease;antiviral therapy for AIDS; and tissue plasminogen activator (TPA) forischemic injury.

XI. Detection of Conditions Involving Altered Apoptosis

IAP polypeptides and nucleic acid sequences find diagnostic use in thedetection or monitoring of conditions involving aberrant levels ofapoptosis. For example, decrease expression of IAP may be correlatedwith enhanced is apoptosis in humans (see section XII, below).Accordingly, a decrease or increase in the level of IAP production mayprovide an indication of a deleterious condition. Levels of LAPexpression may be assayed by any standard technique. For example, IAPexpression in a biological sample (e.g., a biopsy) may be monitored bystandard northern blot analysis or may be aided by PCR (see, e.g.,Ausubel et al., supra; PCR Technology: Principles and Applications forDNA Amplification, H. A. Ehrlich, Ed. Stockton Press, NY; Yap et al.Nucl. Acids. Res. 19:4294, 1991).

Alternatively, a biological sample obtained from a patient may beanalyzed for one or more mutations in the IAP sequences using a mismatchdetection approach. Generally, these techniques involve PCRamplification of nucleic acid from the patient sample, followed byidentification of the mutation (i.e., mismatch) by either alteredhybridization, aberrant electrophoretic gel migration, binding orcleavage mediated by mismatch binding proteins, or direct nucleic acidsequencing. Any of these techniques may be used to facilitate mutant IAPdetection, and each is well known in the art; examples of particulartechniques are described, without limitation, in Orita et al., Proc.Natl. Acad. Sci. USA 86:2766, 1989; Sheffield et al., Proc. Natl. Acad.Sci. USA 86:232, 1989).

In yet another approach, immunoassays are used to detect or monitor IAPprotein in a biological sample. LAP-specific polyclonal or monoclonalantibodies (produced as described above) may be used in any standardimmunoassay format (e.g., ELISA, western blot, or RIA) to measure IAPpolypeptide levels. These levels would be compared to wild-type IAPlevels, with a decrease in LAP production indicating a conditioninvolving increased apoptosis. Examples of immunoassays are described,e.g., in Ausubel et al., supra. Immunohistochemical techniques may alsobe utilized for IAP detection. For example, a tissue sample may beobtained from a patient, sectioned, and stained for the presence of LAPusing an anti-LAP antibody and any standard detection system (e.g., onewhich includes a secondary antibody conjugated to horseradishperoxidase). General guidance regarding such techniques can be found in,e.g., Bancroft and Stevens (Theory and Practice of HistologicalTechniques, Churchill Livingstone, 1982) and Ausubel et al. (supra).

In one preferred example, a combined diagnostic method may be employedthat begins with an evaluation of LAP protein production (for example,by immunological techniques or the protein truncation test (Hogerrorstet al., Nat. Gen. 10:208, 1995)) and also includes a nucleic acid-baseddetection technique designed to identify more subtle IAP mutations (forexample, point mutations). As described above, a number of mismatchdetection assays are available to those skilled in the art, and anypreferred technique may be used. Mutations in IAP may be detected thateither result in loss of IAP expression or loss of LAP biologicalactivity. In a variation of this combined diagnostic method, IAPbiological activity is measured as protease activity using anyappropriate protease assay system (for example, those described above).

Mismatch detection assays also provide an opportunity to diagnose anIAP-mediated predisposition to diseases caused by inappropriateapoptosis. For example, a patient heterozygous for an IAP mutation mayshow no clinical symptoms and yet possess a higher than normalprobability of developing one or more types of neurodegenerative,myelodysplastic or ischemic diseases. Given this diagnosis, a patientmay take precautions to minimize their exposure to adverse environmentalfactors (for example, UV exposure or chemical mutagens) and to carefullymonitor their medical condition (for example, through frequent physicalexaminations). This type of IAP diagnostic approach may also be used todetect IAP mutations in prenatal screens. The IAP diagnostic assaysdescribed above may be carried out using any biological sample (forexample, any biopsy sample or bodily fluid or tissue) in which IAP isnormally expressed. Identification of a mutant IAP gene may also beassayed using these sources for test samples.

Alternatively, an IAP mutation, particularly as part of a diagnosis forpredisposition to LAP-associated degenerative disease, may be testedusing a DNA sample from any cell, for example, by mismatch detectiontechniques. Preferably, the DNA sample is subjected to PCR amplificationprior to analysis.

In order to demonstrate the utility of IAP gene sequences as diagnosticsand prognostics for cancer, a Human Cancer Cell Line Multiple TissueNorthern Blot (Clontech, Palo Alto, Calif.; #7757-1) was probed. Thisnorthern blot contained approximately 2 μg of poly A⁺ RNA per lane fromeight different human cell lines: (1) promyelocytic leukemia HL-60, (2)HeLa cell S3, (3) chronic myelogenous leukemia K-562, (4) lymphoblasticleukemia MOLT-4, (5) Burkitt's lymphoma Raji, (6) colorectaladenocarcinoma SW480, (7) lung carcinoma A549, and (8) melanoma G361. Asa control, a Human Multiple Tissue Northern Blot (Clontech, Palo Alto,Calif.; #7759-1) was probed. This northern blot contained approximately2 μg of poly A⁺ RNA from eight different human tissues: (1) spleen, (2)thymus, (3) prostate, (4) testis, (5) ovary, (6) small intestine, (7)colon, and (8) peripheral blood leukocytes.

The northern blots were hybridized sequentially with: (1) a 1.6 kb probeto the xiap coding region, (2) a 375 bp hiap-2 specific probecorresponding to the 3′ untranslated region, (3) a 1.3 kb probe to thecoding region of hiap-1, which cross-reacts with hiap-2, (4) a 1.0 kbprobe derived from the coding region of bcl-2, and (5) a probe toβ-actin, which was provided by the manufacturer. Hybridization wascarried out at 50° C. overnight, according to the manufacturer'ssuggestion. The blot was washed twice with 2×SSC, 0.1% SDS at roomtemperature for 15 minutes and then with 2×SSC, 0.1% SDS at 50° C.

All cancer lines tested showed increased IAP expression relative tosamples from non-cancerous control tissues (Table 3). Expression of xiapwas particularly high in HeLa (S-3), chronic myelogenous leukemia(K-562), colorectal adenocarcinoma (SW-480), and melanoma (G-361) lines.Expression of hiap-1 was extremely high in Burkitt's lymphoma, and wasalso elevated in colorectal adenocarcinoma. Expression of hiap-2 wasparticularly high in chronic myelogenous leukemia (K-562) and colorectaladenocarcinoma (SW-480). Expression of bcl-2 was upregulated only inHL-60 leukemia cells.

These observations suggest that upregulation of the anti-apoptotic IAPgenes may be a widespread phenomenon, perhaps occurring much morefrequently than upregulation of bcl-2. Furthermore, upregulation may benecessary for the establishment or maintenance of the transformed stateof cancerous cells.

In order to pursue the observation described above, i.e., that hiap-1 isoverexpressed in the Raji Burkitt's lymphoma cell line, RT-PCR analysiswas performed in multiple Burkitt's lymphoma cell lines. Total RNA wasextracted from cells of the Raji, Ramos, EB-3, and Jiyoye cell lines,and as a positive control, from normal placental tissue. The RNA wasreverse transcribed, and amplified by PCR with the following set ofoligonucleotide primers: 5′-AGTGCGGGTTTTTATTATGTG-3′ (SEQ ID NO: 44) and5′-AGATGACCACAAGGAATAAACACTA-3′ (SEQ ID NO: 45), which selectivelyamplify a hiap-1 cDNA fragment. RT-PCR was conducted using a PerkinElmer 480 Thermocycler to carry out 35 cycles of the following program:94° C. for 1 minute, 50° C. for 1.5 minutes, and 72° C. for a minute.The PCR reaction product was electrophoresed on an agarose gel andstained with ethidium bromide. Amplified cDNA fragments of theappropriate size were clearly visible in all lanes containing Burkitt'slymphoma samples, but absent in the lanes containing the normalplacental tissue sample, and absent in lanes containing negative controlsamples, where template DNA was omitted from the reaction (FIG. 17).

XII. Accumulation of a 26 kDa Cleavage Protein in Astrocytoma Cells

A. Identification of a 26 kDa Cleavage Protein

A total protein extract was prepared from Jurkat and astrocytoma cellsby sonicating them (X3 for 15 seconds at 4° C.) in 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 1 mM PMSF, 1 μg/ml aprotinin, and 5 mM benzamidine.Following sonication, the samples were centrifuged (14,000 RPM in amicrofuge) for five minutes. Twenty μg of protein was loaded per well ona 10% SDS-polyacrylamide gel, electrophoresed, and electroblotted bystandard methods to PVDF membranes. Western blot analysis, performed asdescribed previously, revealed that the astrocytoma cell line(CCF-STTG1) abundantly expressed an anti-xiap reactive band ofapproximately 26 kDa, despite the lack of an apoptotic trigger event(FIG. 18). In fact, this cell line has been previously characterized asbeing particularly resistant to standard apoptotic triggers.

A 26 kDa XIAP-reactive band was also observed under the followingexperimental conditions. Jurkat cells (a transformed human T cell line)were induced to undergo apoptosis by exposure to an anti-Fas antibody (1μg/ml).

Identical cultures of Jurkat cells were exposed either to: (1) anti-Fasantibody and cycloheximide (20 μg/ml), (2) tumor necrosis factor alpha(TNF-α, at 1,000 U/ml), or (3) TNF-α and cycloheximide (20 μg/ml). Allcells were harvested 6 hours after treatment began. In addition, as anegative control, anti-Fas antibody was added to an extract after thecells were harvested. The cells were harvested in SDS sample buffer,electrophoresed on a 12.5% SDS polyacrylamide gel, and electroblottedonto PVDF membranes using standard methods. The membranes wereimmunostained with a rabbit polyclonal anti-XIAP antibody at 1:1000 for1 hour at room temperature. Following four 15 minute washes, a goatanti-rabbit antibody conjugated to horse-radish peroxidase was appliedat room temperature for 1 hour. Unbound secondary antibody was washedaway, and chemiluminescent detection of XAP protein was performed. Thewestern blot revealed the presence of the full-length, 55 kDa XIAPprotein, both in untreated and treated cells. In addition, a novel,approximately 26 kDa XIAP-reactive band was also observed in apoptoticcell extracts, but not in the control, untreated cell extracts (FIG.19).

Cleavage of XIAP occurs in a variety of cell types, including othercancer cell lines such as HeLa. The expression of the 26 kDa XIAPcleavage product was demonstrated in HeLa cells as follows. HeLa cellswere treated with either: (1) cyclohexamide (20 μg/ml), (2) anti-Fasantibody (1 μg/ml), (3) anti-Fas antibody (1 μg/ml) and cyclohexamide(20 μg/ml), (4) TNFα (1,000 U/ml), or (5) TNFα (1,000 U/ml) andcyclohexamide (20 μg/ml). All cells were harvested 18 hours aftertreatment began. As above, anti-Fas antibody was added to an extractafter the cells were harvested. HeLa cells were harvested, and thewestern blot was probed under the same conditions as used to visualizexiap-reactive bands from Jurkat cell samples. A 26 kDa XIAP band wasagain seen in the apoptotic cell preparations (FIG. 20). Furthermore,the degree of XIAP cleavage correlated positively with the extent ofapoptosis. Treatment of HeLa cells with cycloheximide or TNFα alonecaused only minor apoptosis, and little cleavage product was observed.If the cells were treated with the anti-Fas antibody, a greater amountof cleavage product was apparent. These data indicate that XIAP iscleaved in more than one cell type and in response to more than one typeof apoptotic trigger.

B. Time Course of Expression

The time course over which the 26 kDa cleavage product accumulates wasexamined by treating HeLa and Jurkat cells with anti-Fas antibody (1μg/ml) and harvesting them either immediately, or 1, 2, 3, 5, 10, or 22hours after treatment. Protein extracts were prepared and western blotanalysis was performed as described above. Both types of cellsaccumulated increasing quantities of the 26 kDa cleavage product overthe time course examined (FIGS. 21A and 21B).

C. Subcellular Localization of the 26 kDa XIAP Cleavage Product

In order to determine the subcellular location of the 26 kDa cleavageproduct, Jurkat cells were induced to undergo apoptosis by exposure toanti-Fas antibody (1 μg/ml) and were then harvested either immediately,3 hours, or 7 hours later. Total protein extracts were prepared, asdescribed above, from cells is harvested at each time point. In order toprepare nuclear and cytoplasmic cell extracts, apoptotic Jurkat cellswere washed with isotonic Tris buffered saline (pH 7.0) and lysed byfreezing and thawing five times in cell extraction buffer (50 mM PIPES,50 mM KCl, 5 mM EGTA, 2 mM MgCl₂, 1 mM DTT, and 20 μM cytochalasin B).Nuclei were pelleted by centrifugation and resuspended in isotonic Tris(pH 7.0) and frozen at −80° C. The cytoplasmic fraction of the extractwas processed further by centrifugation at 60,000 RPM in a TA 100.3rotor for 30 minutes. Supernatants were removed and frozen at −80° C.Samples of both nuclear and cytoplasmic fractions were loaded on a 12.5%SDS-polyacrylamide gel, and electroblotted onto PVDF membranes. Westernblot analysis was then performed using either an anti-CPP32 antibody(Transduction Laboratories Lexington, Ky.; FIG. 22A) or the rabbitanti-XIAP antibody described above (FIG. 22B).

The anti-CPP32 antibody, which recognizes the CPP32 protease (also knownas YAMA or apopain) partitioned almost exclusively in the cytoplasmicfraction. The 55 kDa XIAP protein localized exclusively in the cytoplasmof apoptotic cells, in agreement with the studies presented above, whereXIAP protein in normal, healthy COS cells was seen to localize, byimmunofluorescence microscopy, to the cytoplasm. In contrast, the 26 kDacleavage product localized exclusively to the nuclear fraction ofapoptotic Jurkat cells. Taken together, these observations suggest thatthe anti-apoptotic component of XIAP could be the 26 kDa cleavageproduct, which exerts its influence within the nucleus.

D. In Vitro Cleavage of XIAP Protein and Characterization of theCleavage Product

For this series of experiments, XIAP protein was labeled with ³⁵S usingthe plasmid pcDNA3-6myc-xiap, T7 RNA polymerase, and a coupledtranscription/translation kit (Promega) according to the manufacturer'sinstructions. Radioactively labeled XIAP protein was separated fromunincorporated methionine by column chromatography using Sephadex G-50™.In addition, extracts of apoptotic Jurkat cells were prepared followingtreatment with anti-Fas antibody (1 μg/ml) for three hours. To preparethe extracts, the cells were lysed in Triton X-100 buffer (1% TritonX-100, 25 mM Tris HCl) on ice for two hours and then microcentrifugedfor 5 minutes. The soluble extract was retained (and was labelled“TX100”). Cells were lysed in cell extraction buffer withfreeze/thawing. The soluble cytoplasmic fraction was set aside (andlabelled “CEB”). Nuclear pellets from the preparation of the CEBcytoplasmic fraction were solubilized with Triton X-100 buffer,microcentrifuged, and the soluble fractions, which contains primarilynuclear DNA, was retained (and labelled “CEB-TX100”). Soluble cellextract was prepared by lysing cells with NP-40 buffer, followed bymicrocentrifugation for 5 minutes (and was labeled NP-40). In vitrocleavage was performed by incubating 16 μl of each extract (CEB, TX-100,CEB-TX100, and NP-40) with 4 μl of in vitro translated XIAP protein at37° C. for 7 hours. Negative controls, containing only TX100 buffer orCEB buffer were also included. The proteins were separated on a 10%SDS-polyacrylamide gel, which was then dried and exposed to X-ray filmovernight.

In vitro cleavage of XIAP was apparent in the CEB extract. The observedmolecular weight of the cleavage product was approximately 36 kDa (FIG.23). The 10 kDa shift in the size of the cleavage product indicates thatthe observed product is derived from the amino-terminus of therecombinant protein, which contains six copies of the myc epitope (10kDa). It thus appears that the cleavage product possesses at least twoof the BIR domains, and that it is localized to the nucleus.

XIII. Treatment of HIV Infected Individuals

The expression of hiap-1 and hiap-2 is decreased significantly inHIV-infected human cells. Furthermore, this decrease precedes apoptosis.Therefore, administration of HIAP-1, HIAP-2, genes encoding theseproteins, or compounds that upregulate these genes can be used toprevent T cell attrition in HIV-infected patients. The following assaymay also be used to screen for compounds that alter hiap-1 and hiap-2expression, and which also prevent apoptosis.

Cultured mature lymphocyte CD4⁺ T cell lines (H9, labelled “a”;CEM/CM-3, labelled “b”; 6T-CEM, labelled “c”; and Jurkat, labelled “d”in FIGS. 13A and 13B), were examined for signs of apoptosis (FIG. 13A)and hiap gene expression (FIG. 13B) after exposure to mitogens or HIVinfection. Apoptosis was demonstrated by the appearance of DNA“laddering” upon gel electrophoresis and gene expression was assessed byPCR. The results obtained from normal (non-infected, non-mitogenstimulated) cells are shown in each lane labelled “1” in FIGS. 13A and13B. The results obtained 24 hours after PHA/PMA(phytohemagglutinin/phorbol ester) stimulation are shown in each lanelabelled “2”. The results obtained 24 hours after HIV strain III_(B)infection are shown in each lane labelled “3”. The “M” refers tostandard DNA markers (the 123 bp ladder in FIG. 13B, and the lambdaHindIII ladder in FIG. 13A (both from Gibco-BRL)). DNA ladders (Prigentet al., J. Immunol. Meth., 160:139, 1993), which indicate apoptosis, areevident when DNA from the samples described above are electrophoresed onan ethidium bromide-stained agarose gel (FIG. 13A). The sensitivity anddegree of apoptosis of the four T cell lines tested varies followingmitogen stimulation and HIV infection.

In order to examine hiap gene expression, total RNA was prepared fromthe cultured cells and reverse transcribed using oligo-dT priming. TheRT cDNA products were amplified by PCR using specific primers (as shownin Table 5) for the detection of hiap-2a, hiap-2b and hiap-1. The PCRwas conducted using a Perkin Elmer 480 thermocycler with 35 cycles ofthe following program: 94° C. for one minute, 55° C. for 2 minutes and72° C. for 1.5 minutes. The RT-PCR reaction products wereelectrophoresed on a 1% agarose gel, which was stained with ethidiumbromide. Absence of hiap-2 transcripts is noted in all four cell lines24 hours after HIV infection. In three of four cell lines (all exceptH9), the hiap-1 gene is also dramatically down-regulated after HIVinfection. PHA/PMA mitogen stimulation also appears to decrease hiapgene expression; particularly of hiap-2 and to a lesser extent, ofhiap-1. The data from these experiments is summarized in Table 5. Theexpression of β-actin was consistent in all cell lines tested,indicating that there is not a flaw in the RT-PCR assay that couldaccount for the decrease in hiap gene expression. TABLE 4OLIGONUCLEOTIDE PRIMERS FOR THE SPECIFIC RT-PCR AMPLIFICATION OF UNIQUEIAP GENES Forward Primer Reverse Primer (nucleotide (nucleotide Size ofProduct IAP Gene position*) position*) (bp) h-xiap p2415 (876-896) p2449(1291-1311) 435 m-xiap p2566 (458-478) p2490 (994-1013) 555 h-hiap1p2465 (827-847) p2464 (1008-1038) 211 m-hiap1 P2687 (747-767) p2684(1177-1197) 450 hiap2 p2595 (1562-1585) p2578 (2339-2363)  801^(a) 618^(b) m-hiap2 p2693 (1751-1772) p2734 (2078-2100) 349*Nucleotide position as determined from FIGS. 1-4 for each IAP gene^(a)PCR product size of hiap2a^(b)PCR product size of hiap2b

TABLE 5 APOPTOSIS AND HIAP GENE EXPRESSION IN CULTURED T-CELLS FOLLOWINGMITOGEN STIMULATION OR HIV INFECTION Cell Line Condition Apoptosis hiap1hiap2 H9 not stimulated − + ± PHA/PMA stimulated +++ + ± HIV infected++ + − CEM/CM-3 not stimulated − + ± PHA/PMA stimulated ± + − HIVinfected ± − − 6T-CEM not stimulated − + + PHA/PMA stimulated ± − − HIVinfected + − − Jurkat not stimulated − + ++ PHA/PMA stimulated + + + HIVinfected ± − −XIV. Assignment of Xiap, Hiap-1, and Hiap-2 to Chromosomes Xq25 and11q22-23 by Fluorescence in Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) was used to identify thechromosomal location of xiap, hiap-1 and hiap-2. The probes used werecDNAs cloned in plasmid vectors: the 2.4 kb xiap clone included 1493 bpof coding sequence, 34 bp of 5′ UTR (untranslated region) and 913 bp of3′UTR; the hiap-1 cDNA was 3.1 kb long and included 1812 bp coding and1300 bp of 3′ UTR; and the hiap-2 clone consisted of 1856 bp of codingand 1200 bp of 5′ UTR. A total of 1 μg of probe DNA was labelled withbiotin by nick translation (BRL). Chromosome spreads prepared from anormal peripheral blood culture were denatured for 2 minutes at 70° C.in 50% formamide/2×SSC and subsequently hybridized with the biotinlabelled DNA probe for 18 hours at 37° C. in a solution consisting of2×SSC/70% formamide/10% dextran sulfate. After hybridization, thespreads were washed in 2×SSC/50% formamide, followed by a wash in 2×SSCat 42° C. The biotin labelled DNA was detected by fluoresceinisothiocyanate (FITC) conjugated avidin antibodies and anti-avidinantibodies (ONCOR detection kit), according to the manufacturer'sinstructions. Chromosomes were counterstained with propidium iodide andexamined with a Olympus BX60 epifluorescence microscope. For chromosomeidentification, the slides with recorded labelled metaphase spreads weredestained, dehydrated, dried, digested with trypsin for 30 seconds andstained with 4% Giemsa stain for 2 minutes. The chromosome spreads wererelocated and the images were compared.

A total of 101 metaphase spreads were examined with the xiap probe, asdescribed above. Symmetrical fluorescent signals on either one or bothhomologs of chromosome Xq25 were observed in 74% of the cells analyzed.Following staining with hiap-1 and hiap-2 probes, 56 cells were analyzedand doublet signals in the region 11q22-23 were observed in 83% of cellsexamined. The xiap gene was mapped to Xq25 while the hiap-1 and hiap-2genes were mapped at the border of 11q22 and 11q23 bands.

These experiments confirmed the location of the xiap gene on chromosomeXq25. No highly consistent chromosomal abnormalities involving band Xq25have been reported so far in any malignancies. However, deletions withinthis region are associated with a number of immune system defectsincluding X-linked lymphoproliferative disease (Wu et al., Genomics17:163, 1993).

Cytogenetic abnormalities of band 11q23 have been identified in morethan 50% of infant leukemias regardless of the phenotype(Martinez-Climet et al., Leukaemia 9:1299, 1995). Rearrangements of theMLL Gene (mixed lineage leukemia or myeloid lymphoid leukemia; ZieminVan der Poel et al., Proc. Natl. Acad. Sci. USA 88:10735, 1991) havebeen detected in 80% of cases with 11q23 translocation, however patientswhose rearrangements clearly involved regions other than the MLL genewere also reported (Kobayashi et al., Blood 82:547, 1993). Thus, the IAPgenes may follow the Bcl-2 paradigm, and would therefore play animportant role in cancer transformation.

XV. Preventive Anti-Apoptotic Therapy

In a patient diagnosed to be heterozygous for an IAP mutation or to besusceptible to IAP mutations (even if those mutations do not yet resultin alteration or loss of IAP biological activity), or a patientdiagnosed as HIV positive, any of the above therapies may beadministered before the occurrence of the disease phenotype. Forexample, the therapies may be provided to a patient who is HIV positivebut does not yet show a diminished T cell count or other overt signs ofAIDS. In particular, compounds shown to increase IAP expression or IAPbiological activity may be administered by any standard dosage and routeof administration (see above). Alternatively, gene therapy using an IAPexpression construct may be undertaken to reverse or prevent the celldefect prior to the development of the degenerative disease.

The methods of the instant invention may be used to reduce or diagnosethe disorders described herein in any mammal, for example, humans,domestic pets, or livestock. Where a non-human mammal is treated ordiagnosed, the IAP polypeptide, nucleic acid, or antibody employed ispreferably specific for that species.

Other Embodiments

In other embodiments, the invention includes any protein which issubstantially identical to a mammalian IAP polypeptides (FIGS. 1-6; SEQID NOs: 1-42); such homologs include other substantially purenaturally-occurring mammalian IAP proteins as well as allelic variants;natural mutants; induced mutants; DNA sequences which encode proteinsand also hybridize to the IAP DNA sequences of FIGS. 1-6 (SEQ ID NOS:1-42) under high stringency conditions or, less preferably, under lowstringency conditions (e.g., washing at 2×SSC at 40° C. with a probelength of at least 40 nucleotides); and proteins specifically bound byantisera directed to a IAP polypeptide. The term also includes chimericpolypeptides that include a IAP portion.

The invention further includes analogs of any naturally-occurring IAPpolypeptide. Analogs can differ from the naturally-occurring IAP proteinby amino acid sequence differences, by post-translational modifications,or by both. Analogs of the invention will generally exhibit at least85%, more preferably 90%, and most preferably 95% or even 99% identitywith all or part of a naturally occurring IAP amino acid sequence. Thelength of sequence comparison is at least amino acid residues,preferably at least 25 amino acid residues, and more preferably morethan 35 amino acid residues. Modifications include in vivo and in vitrochemical derivatization of polypeptides, e.g., acetylation,carboxylation, phosphorylation, or glycosylation; such modifications mayoccur during polypeptide synthesis or processing or following treatmentwith isolated modifying enzymes. Analogs can also differ from thenaturally-occurring IAP polypeptide by alterations in primary sequence.These include genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual(2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included arecyclized peptides, molecules, and analogs which contain residues otherthan L-amino acids, e.g., D-amino acids or nonnaturally occurring orsynthetic amino acids, e.g., β or γ amino acids. In addition tofull-length polypeptides, the invention also includes IAP polypeptidefragments. As used herein, the term “fragment,” means at least 20contiguous amino acids, preferably at least 30 contiguous amino acids,more preferably at least 50 contiguous amino acids, and most preferablyat least 60 to 80 or more contiguous amino acids. Fragments of IAPpolypeptides can be generated by methods known to those skilled in theart or may result from normal protein processing (e.g., removal of aminoacids from the nascent polypeptide that are not required for biologicalactivity or removal of amino acids by alternative mRNA splicing oralternative protein processing events).

Preferable fragments or analogs according to the invention are thosewhich facilitate specific detection of a IAP nucleic acid or amino acidsequence in a sample to be diagnosed. Particularly useful LAP fragmentsfor this purpose include, without limitation, the amino acid fragmentsshown in Table 2.

1. A substantially pure polypeptide comprising an amino acid sequence atleast 85% identical to SEQ ID NO:32.
 2. The polypeptide according toclaim 1, wherein the amino acid sequence is at least 90% identical toSEQ ID NO:32.
 3. The polypeptide according to claim 1, wherein the aminoacid sequence is at least 95% identical to SEQ ID NO:32.
 4. Asubstantially pure polypeptide comprising the sequence of SEQ ID NO:32.5. A substantially pure polypeptide comprising the amino acid sequenceof which comprises residues 560-605 of SEQ ID NO:8.
 6. The polypeptideaccording to claim 5, wherein the amino acid sequence is at least 85%identical to residues 560-605 of SEQ ID NO:8.
 7. The polypeptideaccording to claim 5, wherein the amino acid sequence is at least 90%identical to residues 560-605 of SEQ ID NO:8.
 8. The polypeptideaccording to claim 5, wherein the amino acid sequence is at least 95%identical to residues 560-605 of SEQ ID NO:8.
 9. A substantially purepolypeptide the amino acid sequence of which consists of SEQ ID NO:32.10. A substantially pure polypeptide comprising an amino acid sequenceat least 85% identical to SEQ ID NO:8, wherein the polypeptide comprisesthe sequenceGlu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,where Xaa1 is any amino acid, Xaa2 is Glu or Asp, Xaa3 is Val or Ile(SEQ ID NO:1).
 11. The polypeptide according to claim 10, wherein theamino acid sequence is at least 90% identical to SEQ ID NO:8.
 12. Thepolypeptide according to claim 10, wherein the amino acid sequence is atleast 95% identical to SEQ ID NO: 8.